The studies proposed here continue our work on the molecular mechanism of sensory transduction by hair cells. A great deal of physiological evidence acquired over the last ten years indicates that the transduction channels in vertebrate hair cells are directly activated by mechanical stimuli: that the tip links on stereocilia convey the displacement of the hair bundle to the channels, and that the stress causes channels to open. Similarly, physiological evidence suggests that adaptation of the transduction channels in the presence of a steady displacement comes about by a decline in the stress reaching channels, most likely by a movement of the tip link attachment. A particular myosin, type Ibeta, has been cloned from hair cells; it is thought to be the motor protein that drives this movement. In this study, we will investigate three aspects of the transduction apparatus in bullfrog hair cells. First, we will determine whether the mechanically sensitive transduction channels are at the upper or lower (or both) ends of the tips links, using electrophysiological recording from single hair cells. The molecular identity of the channel is unknown, and knowledge of its specific location will help in testing candidates for the channel protein. Second, we will test the hypothesis that a myosin is the adaptation motor protein, in several ways. We will see if antibodies to the myosin Ibeta, VI or VII tail, or the tail domain itself, bind to the upper insertion of the tip link. We will also see if either antibodies or the tail itself interfere with adaptation when measured physically. If myosin Ibeta can be shown to be the motor, this will be the first identified protein element of the transduction apparatus, and it may lead us to other components. Moreover, methods that we develop to test the role of this protein in transduction may be used to test other candidate proteins when identified. Third, we will identify proteins that bind to the myosin tail using molecular biological screening of a "two hybrid" library in yeast. These proteins could be additional linker proteins, they could be part of the tip link, or they could be subunits of the transduction channel. This understanding of hair bundle structures and the proteins associated with transduction may elucidate certain pathological conditions of the auditory system. For instance, one of the myosins we cloned from hair cells is defective in Usher Syndrome, the most common inherited deafness in humans. Identification of the motor protein and proteins that bind to it may provide genetic tests for other inherited deafnesses.